Construction of chromosomal integrants of Bacillus sphaericus 2362 by conjugation with Escherichia coli

IncP-based plasmids conjugated between Escherichia coli and mosquitocidal s trains of Bacillus sphaericus at frequencies of 10(-7) to 10(-9) per recipi ent. Plasmid transfer was most efficient when a restriction-deficient strai n of B. sphaericus 2362 (serotype 5a5b) was used as recipient and was least efficient with recipients from serotypes 1a and 2a2b. A deleted version of the cryptic locus 'gene 80' from strain 2362 was cloned into the suicide v ector pMTL30, which could not replicate in B. sphaericus to provide a site for chromosomal integration. Conjugational transfer from E. coli and integr ation into the B. sphaericus recipient chromosome was achieved with this co nstruct. The coding region of the cry11A gene from Bacillus thuringiensis s ubsp, israelensis was PCR-amplified and fused to the promoter of the crysta l protein (Bin) gene of B. sphaericus 2362. This construct was cloned into the integrative vector, conjugated with B. sphaericus 2362 and chromosomal integrants were recovered which harboured the cry11A gene. The fusion gene was efficiently transcribed in the recombinant host, but cells failed to ac cumulate appreciable amounts of Cry11A toxin. This system offers a simple a nd efficient means of transferring plasmids into B. sphaericus and obtainin g chromosomal integration for strain construction and gene analysis. (C) 20 00 Editions scientifiques et medicales Elsevier SAS.